Protease enzymes are involved in a variety of biological phenomena, for example, in protein activation and cell signalling. Protease activity plays a key role in processes such as blood clotting, apoptosis and hormone regulation. Proteases are also essential to the function of a variety of viral and microbial pathogens. There is an increasing interest in the development of protease inhibitors for use as therapeutic agents.
The determination of protease activity in biological samples is important in the analysis of processes such as apoptosis, in the screening of potential protease inhibitors and in the monitoring of sample purity, for example during protein purification. Protease enzymes hydrolyse amides and esters to produce peptides, single amino acids or labelled amino acid fragments depending on the structure of the substrate and the nature of the enzyme. The determination of protease activity may be performed using either a naturally occurring protein substrate or a synthetic peptide substrate analogue that is labelled, for example with a fluorophore or a chromophore. The determination of protease activity may also be performed using a labelled short synthetic peptide that optionally incorporates a protease recognition sequence. In some cases the determination of protease activity may be performed using a labelled single amino acid. In these cases protease activity is detected by the ability of the protease to cleave the bond between the single amino acid and the marker with which it is labelled.
The nature of the protein used depends on the solubility required and the particular requirements of the assay; bovine serum albumin (BSA) or casein are commonly used. Gelatin, ovalbumin and cross-linked proteins have also been used.
Most detection assays on the market use a labelled substrate analogue. The detection of protease activity can be performed using a homogenous or a heterogenous reaction set up. In a homogenous detection assay, the substrate is typically in solution and the product is also in solution. Fluorescence-based detection systems using fluorophores (for example fluoroscein, rhodamine or BODIPY fluorophores) generally operate according to one of two principles: detection of a fluorescent signal following cleavage of a multiply labelled self-quenched protein (ENZCHEK protease assay kits, Molecular Probes Inc.), or detection of a change in the size of the fluorescent-labelled moiety/conjugate using fluorescence polarization techniques (BEACON protease activity detection kit, PanVera Corporation, ENZCHEK polarization kit, Molecular Probes Inc.).
In one commonly practised method, a peptide substrate is used that is labelled at the carboxyl terminus with a dye possessing an amine functionality. Such a dye may be a chromophore or a fluorophore, for example coumarin, fluoroscein, rhodamine or BODIPY (Molecular Probes Inc, APOALERT, CPP32 protease assay kits, Clontech). The amide bond that couples dye and amino acid is cleaved by the protease to produce an amine derivative. That change in structure affects the spectral characteristics of the dye and a detectable signal is thus produced. An alternative strategy for homogenous detection is to use a peptide substrate that is labelled at one side of a cleavage site with a donor fluorophore and at the other side with an acceptor quencher, which together form a fluorescence resonance energy transfer (FRET) pair. Cleavage of the peptide results in separation of the donor and acceptor and therefore produces a change in fluorescence signal. Hydrolysis of a specific peptide sequence can be detected by a gel based analysis (PEPTAG protease assay, Promega).
A heterogenous assay typically involves the cleavage of a dye-labelled fragment of an immobilised substrate and subsequent analysis of the liquid phase (PROTEASESPOTS, Jerini AG, PROCHECK Universal Protease Assay, Intergen).
Protease activity may also be determined using an unmodified (i.e. naturally occurring) protein substrate. Assays using unmodified protein substrates generally require precipitation of the undigested substrate and subsequent detection of cleaved protein fragments. Such detection can be, for example, by measurement of absorbance at 278 nm, or by detection of the resulting primary amine functionality. Such methods often lack sensitivity, require sampling to obtain kinetic data and depend on quantitative precipitation for accurate results. Alternatively, hydrolysis of succinylated proteins may be detected following reaction of the resulting peptide fragments with TNBSA (trinitrobenzenesulfonic acid).
Except where the contrary is apparent from the context, the term “substrate” is used throughout the remainder of this document to include both naturally occurring substrates and synthetic substrates. Synthetic substrates include synthetic analogues of naturally occurring substrates, synthetic peptides incorporating a protease recognition sequence, and other synthetic peptides, and single amino acids. Single amino acids are regarded as a substrate because, although they lack an internal bond capable of being cleaved by a protease enzyme, such a bond may be formed through the attachment of a marker.
The term “protease” as used herein is intended to include within its scope proteins that are known as proteinases.
The term “peptide” and “protein” are used interchangeably herein and both include amino acid sequences of any length including those with a small number of amino acid residues, for example five residues or three residues. The terms “peptide” and “protein” include both molecules made in cells and molecules made by cell-free synthesis. The terms “peptide” and “protein” include molecules having naturally occurring, semi-synthetic or artificial sequences, which sequences may include amino acids that do not occur naturally in proteins. For example, the terms “peptide” and “protein” refer to an amino acid sequence of a recombinant or non-recombinant peptide having an amino acid sequence of (i) a native peptide, (ii) a biologically active fragment of a native peptide, (iii) a biologically active peptide analogue of a native peptide, (iv) a biologically active variant of a native peptide, (v) a peptide having an artificial sequence comprising a biologically active consensus sequence, or (vi) a peptide having a wholly artificial sequence.
The term “amino acid” includes naturally occurring amino acids and amino acids that do not occur naturally in proteins. It also includes amino acid derivatives, for example acylated amino acids, protease enzymes, hydrolyse amides, and esters. It will therefore be understood that a key requirement of a labelled protease substrate is the presence of bonds capable of being hydrolysed by protease enzymes. In general, therefore, the terms “protein”, “peptide” and “amino acid” are to be interpreted broadly to encompass any derivative molecules that provide one or more bonds capable of being hydrolysed by protease enzymes. These hydrolysable bonds may be provided within the structure of the molecule itself or alternatively or additionally may be formed when the molecule is labelled by the attachment of a marker.